The bacterial cell wall plays a fundamental role in several critical biological processes, including protecting microorganisms against lysis by osmotic pressure and contributing to subcellular organization and overall morphology. Fragments of peptidoglycan, the structural backbone of the wall, function as toxins toward humans and animals and are targeted by the innate immune response in these higher organisms. In addition, by imparting to bacteria their different shapes, the wall creates polarities, influences bacterial differentiation, and contributes directly or indirectly to nutrient accumulation, attachment to surfaces, motility, chromosomal segregation, predator avoidance, biofilm formation, and pathogenesis. Thus, this field addresses concerns that are both basic and practical: basic, in considering how cells are constructed; and practical, because these characteristics contribute to bacterial virulence and determine the effectiveness of antibiotic therapy. The long-term goal of this project is to understand the structure, synthesis, regulation and functional implications of peptidoglycan and the enzymes that create and modify it. More specifically, we wish to know how cells differentiate their walls into structural and functional domains, how they process the wall during cell division, and how they mold it to produce cells of different shapes. Recent discoveries demonstrate that the premier cell division protein, FtsZ, does far more than its acknowledged role in initiating and directing cell division. First, division relies on the activities of peptidoglycan-modifying enzymes, and this relationship, when disturbed, can lead to cell dissolution. Second, it is now apparent that FtsZ directs cell wall synthesis throughout the cell cycle: diffusely into the sidewalls as bacteria elongate; in a concentrated narrow band at the cell center prior to the initiation of septation; and during the constriction phase of cell division. We propose to extend and refine our understanding of these aspects of bacterial physiology by pursuing the following specific aims: 1) examining more closely the mechanisms that drive FtsZ-dependent peptidoglycan synthesis, 2) characterizing new phenotypes associated with FtsZ, and 3) determining the structure of the cell wall. These goals will be realized by defining the proteins that, along with FtsZ, are responsible for the different types of peptidoglycan synthesis, by isolating and characterizing mutations in pathways that intersect with FtsZ and cell wall synthesis, and by creating new tools to examine the chemical nature of the wall. Understanding the processes underlying cellular integrity, cell shape and division will shed light on the genesis, structure and topological regulation of peptidoglycan synthesis and on prokaryotic morphology in general. PUBLIC HEALTH RELEVANCE: A rigid cell wall is essential for bacterial survival, and a large number of our most important antibiotics inhibit the proteins that create this protective structure. Because antibiotic resistance is increasing, it has become especially urgent to understand completely how the bacterial wall is made, the activities of the proteins that make it, and what its final structure looks like. This project will approach this goal by discovering ways in which proteins interact to synthesize the cell wall and by creating new tools for visualizing its molecular composition. [unreadable] [unreadable] [unreadable]